RG17PE38, a novel immunotoxin target to gastric cancer with overexpressed CCK-2R. (May 2013)

• Record Type: Journal Article
• Authors: Song, Jie; Ren, Honglin; Li, Yansong; Xu, Junyan; Kong, Hong; Tong, Weihua; Zhou, Yu; Gao, Shiqi; Liu, Yanyan; Hui, Qi; Peng, Qisheng; Lu, Shiying; Liu, Zengshan
• Is Part Of: Journal of drug targeting. Volume 21:Number 4(2013)
• Page Start: 375
• Page End: 382
• Publication Date: 2013-05
• Subjects: Drug delivery systems -- Periodicals; Drug Delivery Systems; Vehicles; Drug Administration Routes; Drug Evaluation; 615.7
• DOI: 10.3109/1061186X.2012.757770
• Languages: English
• ISSNs: 1061-186X

Abstract

<abstract> <title>Abstract</title> <p> <italic>Background</italic>: Gastrin/cholecystokinin subtype 2 receptor (CCK2R) is overexpressed in several types of tumors. Gastrin-17 (G17) peptide has a high affinity with CCK2R. These characters suggest that G17 may be useful for target cancer therapy.</p> <p> <italic>Purpose</italic>: Construct a new immunotoxin (IT) targeting of CCK2R overexpressed gastric cancer.</p> <p> <italic>Methods</italic>: Two ITs were generated using forward and reverse G17 peptides fused with PE38. To get a high yield, codon optimized gene and optimized fermentation parameters were used in large-scale protein expression. An immunoaffinity technique was introduced into pseudomonas exotoxin (PE)-derived IT purification procedure. G17 competition, GST pull-down and indirect immunoflourescence assays were carried out to confirm the interaction between rG17 and CCK2R. Then, several cytotoxic assays were carried out on 18 cell lines, and an <italic>in vivo</italic> antitumor activity experiment was tested in nude mice.</p> <p> <italic>Results</italic>: The rG17PE38 showed specific cytotoxicity on three gastric cancer cells, while G17PE38 did not. After optimization, the expression level reached about 40% in medium deprived of NaCl. Next, 15–27.5 mg of pure rG17PE38 per 1 L of cultures was obtained. Results of G17 competition, GST pull-down and indirect immunoflourescence assays demonstrated that rG17 have a specific interact with CCK2R. Purified rG17PE38 showed high cytotoxicity on gastric cancer cell lines with the IC<sub>50</sub> value of 0.6–4 ng · mL<sup>−1</sup>. Treatment of nude mice inoculated with BGC-823 tumor xenografts with rG17PE38 efficiently inhibited tumor size.</p> <p> <italic>Conclusions and discussion:</italic> The present study demonstrates that reversed G17 could be used as target moiety of PE-derived IT and the rG17PE38 could be developed as a new immunotherapy agent. Codon optimized gene could increase the rG17PE38 expression level in <italic>E. coli</italic> and furthermore NaCl inhibits the rG17PE38 expression in large scale. Meanwhile, our present study inducts an immunoaffinity method in the IT purification procedure, which could purify the PE-derived ITs in native form.</p> </abstract>